AU692817B2 - Construction of (pasteurella haemolytica) vaccines - Google Patents

Abstract

Methylation of DNA can be a critical step in the introduction of DNA into P. haemolytica. A methyltransferase has been isolated and molecularly cloned for this purpose. Use of the methyltransferase has allowed construction of defined, attenuated aroA mutants for use as vaccines to protect cattle.

Description

~ILbPaO~Bn~ lb WO 95/16045 PC'IUS94/14095 CONSTRUCTION OF PASTEURELLA HAEMOLYTICA VACCINES TECHNICAL AREA OF THE INVENTION The invention relates to the area of bacterial genetic engineering. In particular, it relates to the bacteria Pasteurella haemolytica.

BACKGROUND OF THE INVENTION The microorganism P. haemolytica biotype A, serotype 1, is the principal causative agent of pneumonic pasteurellosis in cattle. If techniques could be developed for introducing exogenous DNA into P. haemolytica, it would be possible to produce site-specific mutations in this bacterium. Such mutants could provide "rationally" attenuated strains for use as live vaccines.

Attenuated auxotrophic mutants were first described by Bacon and Burrows in the early 1950's. They reported that attenuated auxotrophs of Salmonella typhi defective in the aromatic amino acid biosynthetic pathway were avirulent in mice.

Subsequently, it has been demonstrated in widely diverse bacteria that disrupting the aromatic amino acid biosynthetic pathway produces attenuated organisms. For example, attenuated strains of the invasive bacteria Salmonella typhi, Salmonella typhimurium, Shigella flexneri, and Yersina enterocolitica, were generated by introducing mutations in their respective aroA genes. Also attenuation was produced in the non-invasive bacteria Bordetella pertussis and Pasteurella multocida through aroA inactivation. Strains which carry aroA mutations are unable to synthesize chorismic acid from which p-aminobenzoic acid, dihydrobenzoate, and aromatic amino acids are produced. It is likely that the absence of one or more of these compounds in vivo is responsible for the poor growth of aroA mutants in the hosts.

I P~ I Live attenuated bacterial strains generally provide superior protection as compared to killed bacterial vaccines (bacterins). In general, live vaccines elicit a stronger cell mediated response in the host than do bacterins. The superior immunity provided by attenuated live organisms may be explained by their ability to induce expression of stressproteins and, possibly, of certain toxins within the host. The immune response generated by live organisms would be directed against these abundant proteins and thereby provide better protection.

There is a long-felt and continuing need in the art for veterinary vaccines to protect cattle from P. haemolytica infection. There also is a need for techniques for introducing DNA into P. haemolytica.

SUMMARY OF THE INVENTION In a first aspect, the invention provides a method for mutagenizing P. haemolytica.

9 In a second aspect, the invention provides a P. haemolytica gene for production of an enzyme for use in preparing genetic material for introduction into P. haemolytica.

0* 9.

S 15 In a third aspect, there is provided an enzyme for use in preparing genetic material for introduction into P. haemolytica.

In a fourth aspect, the invention provides a plasmid for unstable introduction of genetic material P. haemolytica.

A fifth aspect of the invention are P. haemolytica mutant strains.

20 In a sixth aspect of the invention, there is provided a live, attenuated vaccine against P. haemolytica infection.

In a seventh aspect, a genetically engineered P. haemolytica is provided.

These and other aspects of the invention are provided by one or more of the embodiments described below. In one embodiment of the invention a method for sitedirected mutagenesis of P. haemolytica is provided. The method comprises the steps of: isolating a DNA region from P. haemolytica in which region a mutation is desired; introducing a mutation into said DNA region to form a mutated DNA region; methylating said mutated DNA region with a methylating FHPMELCD\98121019.4- 5 May 1998(10:58) -t kc~ J PCT/US94/14095 WO 95116045 -3enzyme, to form methylated DNA, which methylated DNA is refractory to endonuclease cleavage at GATGC and GCATC sequences; introducing said methylated DNA into P. haemolytica to form transformants; and screening said transformants for those which have said mutation in said region on chromosomal DNA of said P. haemolytica cell.

In an alternative embodiment of the invention site-directed mutagenesis of P. haemolytica is accomplished by the steps of: isolating a DNA region from P.

haemolytica in which region a mutation is desired; introducing a mutation into said DNA region to form a mutated DNA region; introducing said methylated DNA into a P. haemolytica cell which does not express a PhaI restriction endonuclease, to form transformants; and screening said transformants for those which have said mutation in said region on chromosomal DNA of said P. haemolytica cell.

In another embodiment of the invention an isolated and purified gene is provided. The gene encodes PhaI methyltransferase.

In still another embodiment of the invention another isolated and purified gene is provided. The gene encodes PhaI restriction endonuclease.

In yet another embodiment of the invention a preparation of PhaI methyltransferase is provided. The preparation is free from PhaI restriction endonuclease.

In still another embodiment of the invention a preparation of PhaI restriction endonuclease is provided. The preparation is free from PhaI methyltransferase.

In another embodiment of the invention a chimeric plasmid is provided which is suitable for unstable introduction of genetic material into P. haemolytica.

The plasmid comprises a 4.2 kb P. haemolytica plasmid encoding a streptomycin resistance determinant deposited at the American Type Culture Collection as Accession No. ATCC 69499; and a plasmid which cannot replicate in P.

haemolytica.

In an additional embodiment of the invention a P. haemolytica mutant is provided. The mutant is made by the process of the invention described in more detail below.

1 3 PP r WO 95/16045 PCTIUS94/14095 -4- In another embodiment of the invention a P. haemolytica mutant is provided which does not express the PhaI restriction endonuclease.

In another embodiment of the invention a P. haemolytica aroA mutant is provided.

In still another embodiment of the invention a vaccine is provided. The vaccine comprises an attenuated, live, mutant of P. haemolytica which has an aroA mutation.

In yet another embodiment of the invention an isolated and purified P. haemolytica strain is provided. The strain has been genetically modified by the introduction of DNA.

These and other embodiments of the invention provide the art with the means to construct desirable mutants of the economically important and previously intractable pathogen P. haemolytica.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Determination of Phal cleavage positions alongside that of SfaNI.

Lanes 1 and 3 cut with PhaI; lanes 2 and 4 cut with SfaNI. The cleavage products of Phal and SfaNI migrated 0.5 bp faster than the corresponding sequence bands because the labeled primer for extension had a 5' phosphate, whereas the primer for sequencing did not (Juarin et al., Gene 39:191-201 (1985)).

Figure 2. Protection against PhaI digestion by cloned Phalmethyltransferase. Lanes 1 and 2 plasmid pPhAAroACmR-pD80 from E. coli incubated without and with Phal. Lanes 3 and 4 plasmid pPhAAroACm

R

from E. coli PhaIMtase incubated without and with PhaI.

Figure 3. Southern blot analysis of P. haemolytica strain DNA digested with EcoRI lane 1, ClaI lane 2, PstI lane 3, or HindIl lane 4. The membrane was hybridized with an E. coli aroA probe and washing was performed under low-stringency conditions.

Figure 4. Nucleotide sequence and deduced amino acid sequence of P.

haemolytica aroA.

Figure 5. Construction of a P. haemolytica aroA mutant. The hybrid plasmid pPharoA'AmpRpD70 was successfully used to produce an aroA mutant.

L II _UU~ WO 95/16045 PC/'IUS94/140O95 Figure 6. Southern hybridization of genomic DNAs from the parental strain, P. haemolytica strain NADC-D60, the aroA mutant, and P. haemolytica strain NADC-D70 and the hybrid plasmid pPharoA-AmpRpD70. All the DNAs used in the blots shown here were digested with HindIII. Figure 6A. Lanes: 1, P. haemolytica strain NADC-D60; 2, aroA mutant; 3, pPharoA'AmpRpD70 probed with P. haemolytica aroA. Figure 6B. Lanes: 1, P. haemolytica strain NADC- 2, aroA mutant; 3, pPharoA'Amp'pD70 probed with P. haemolytica AmpR fragment. Figure 6C. Lanes: 1, P. haemolytica strain NADC-D70; 2, aroA mutant; 3, pPharoA-Amp'pD70 probed with P. haemolytica AmpR plasmid.

Figure 6D. 1, P. haemolytica strain NADC-D60; 2, aroA mutant; 3, pPharoA" probed with pBCSK. DNA was isolated from P. haemolytica strain and run in Lane 1 of blot B to demonstrate that if plasmid DNA was present in the bacteria it would also be present in our DNA preparations.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS It is a discovery of the present invention that P. haemolytica contains at least one restriction-modification system, called herein the PhaI system. Both the restriction endonuclease and the methyltransferase have been molecularly cloned.

One such molecular clone coli PhaIMtase) has been deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD, 20852, USA, on December 2, 1993, under the terms of the Budapest Treaty as Accession No.

ATCC 69500. A preliminary sequence of the methyltransferase gene has been determined. The predicted amino acid sequence of the methyltransferase contains sequence motifs which are consistent with an adenine-methylating specificity.

Provided with the molecular clone of PhalMtase (Accession No. ATCC 69500) one of ordinary skill in the art can readily obtain a preparation of either or both enzymes free of other P. haemolytica proteins. A lysate of a non-P.

haemolytica bacterium carrying one of the cloned enzymes would provide such a preparation. If one desires a preparation of each of the enzymes free of the other enzyme, one of skill in the art can readily subclone to separate the two genes. The methyltransferase gene has been cloned into a plasmid which when introduced into a cell produces PhaI methyltransferase but is free of the PhaI restriction p -a 91 CI 1II Ln Oi/.i\A PCTUS94/14095 -6endonuclease. The PhaI restriction endonuclease gene can be cloned on a plasmid free of the methyltransferase gene by introduction of the cloned gene into host cells which express either the PhaI or the SfaNI methyltransferase.

Provided with PhalMtase (ATCC Accession No. ATCC 69500) one of skill in the art can also readily obtain an isolated and purified gene encoding either or both the PhaI restriction and methyltransferase enzymes. Standard techniques, such as cesium chloride gradients, phenol and chloroform extractions, etc., can be used for purifying plasmid DNA from the deposited E. coli bacteria. The genes can be isolated together from the deposited bacteria, or they can be subcloned, as discussed above, to isolate the two genes from each other.

It has also been discovered by the present inventors, that a barrier to transformation of P. haemolytica can be overcome by treating DNA with a methylating enzyme, such as the PhaI methyltransferase. Such enzymes modify DNA substrates such that endonucleases which recognize 5'-GATGC-3' or GCATC-3' sequences are inhibited in their ability to digest such modified substrates. Examples of such endonucleases are PhaI endonuclease and SfaNI endonuclease. While applicants do not wish to be bound by any particular hypothesis on the mechanism of action of such methyltransferase enzymes, it appears that the PhaI methyltransferase methylates specific adenine residues in

DNA.

Methylation of DNA substrates for transformation (electroporation, or other means of introduction of DNA into cells) can be accomplished in vitro or in vivo.

For in vitro methylation, DNA is incubated with a preparation of methyltransferase in the presence of a methyl donor, such as S-adenosylmethionine (SAM). In vivo methylation can be accomplished by passaging the DNA substrate through a bacterium which contains an appropriate methyltransferase, such as Phal or SfaNI methyltransferase. A mutant or natural variant of P. haemolytica which lacks the Phal endonuclease could also be used to prepare DNA for subsequent introduction into P. haemolytica. Such a mutant can be made inter alia according to the method for site-directed mutagenesis disclosed herein.

WO 95/16045 I'CT/US94/14095 -7- Site-directed mutagenesis of P. haemolyrica can be accomplished according to the present invention by first isolating a wild-type DNA region from P.

haemolytica. As described below in the examples, an aroA gene can be isolated using aroA DNA from other bacteria as hybridization probes. The sequence of the P. haemolytica aroA gene is shown in SEQ ID NO. 1. Similarly other genes can be isolated from P. haemolytica. Another desirable gene for mutations is the PhaI endonuclease gene, which is provided in PhaIMtase (ATCC Accession No.

ATCC 69500). Other genes in which mutations may be desirable are genes in the leukotoxin operon A, B, D) and neuraminidase. A mutation is created in the isolated, wild-type DNA region according to any method known in the art. For example, the isolated DNA can be chemically mutagenized, either in a bacterium or in vitro. Alternatively, restriction endonucleases can be used to create precise deletions or insertions in vitro. Other methods as are known in the art can be used as is desirable for a particular application.

After P. haemolytica DNA has been isolated and mutagenized, it is methylated as described above. Then it can be introduced into P. haemolytica according to any technique known in the art, including but not limited to transfection, transformation, electroporation, and conjugation. Alternatively, rather than methylating the mutagenized DNA and introducing it into a P.

haemolytica which expresses PhaI restriction endonuclease, one can omit the methylation of the mutagenized DNA and introduce the mutagenized DNA into a P. haemolytica cell which does not express the PhaI restriction endonuclease.

Such cells can be isolated from nature by extensive screening, isolated following chemical mutagenesis of a cell which does express the PhaI restriction endonuclease, or made by the site-directed mutagenesis method disclosed herein.

According to one aspect of the invention, the mutagenized and methylated P. haemolytica DNA region is introduced into a P. haemolytica cell on a plasmid which includes a P. haemolytica approximately 4.2 kb streptomycin resistance determining plasmid (pD70). This plasmid has also been deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD, 20852, USA, on December 2, 1993, under the terms of the Budapest Treaty as Accession IPIB~I~R WO 95/16045 PCT/US94/14095 -8- No. ATCC 69499. While applicants do not wish to be bound by any particular theory, it appears that the pD70 streptomycin resistance determining plasmid allows the introduced DNA to be replicated and maintained, albeit unstably, for a period of time sufficient to allow gene conversion (replacement of the chromosomal copy of the gene with the introduced mutant copy of the gene) to occur. Gene conversion can be monitored inter alia by Southern hybridization with probes to the gene of interest, by screening for genetic markers on the introduced DNA construct (such as ampicillinR or streptomycinR), and by screening for the presence/absence of plasmid in the transformed cells' progeny.

A chimeric plasmid, as described above, is provided which is suitable for the unstable introduction of DNA into P. haemolytica. The chimeric plasmid comprises the approximately 4.2 kb streptomycin resistance determining plasmid, as well as a plasmid which cannot replicate in P. haemolytica but can replicate in another cell type. To use such a chimeric plasmid, typically a region of the chromosome of P. haemolytica which has been mutagenized is ligated into the plasmid. Maintenance of the chimeric plasmid in P. haemolytica can be selected, for example by using an appropriate antibiotic to which the plasmid confers resistance. After a selected number of generations, antibiotic selection can be removed, and the cells tested to determine whether the introduced region of P.

haemolytica has replaced the genomic copy.

Also provided by the present invention are mutant strains made by the disclosed method of site-directed mutagenesis. One such mutant aroA') has been deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD, 20852, USA, on December 2, 1993, under the terms of the Budapest Treaty as Accession No. ATCC 55518. Such mutants can provide the veterinary arts with attenuated, live strains of P. haemolytica which are suitable for vaccines to induce protective immunity against P. haemolytica infection. For vaccine production, it is desirable that the mutation which attenuates the P. haemolytica be an essentially non-reverting mutation. Typically these are deletion or insertion mutations, the latter not being caused by a transposable element. Strains which contain multiple attenuating mutations may P "I ~eQq llls also be used, so that the risk of reversion to a wild-type, virulent P. haemolytica is vanishingly small.

Another mutant strain which can be made by the site-directed mutagenesis method disclosed is one which is a PhaI restriction endonuclease negative. Such a strain is useful for genetic engineering in P. haemolytica. Such a strain can be a recipient of DNA which is not PhaI methyltransferase methylated, yet would yield DNA which is Phal methyltransferase methylated.

The present invention thus allows those of ordinary skill in the art to stably introduce DNA into P. haemolytica. The DNA can be from other strains or species. The S 10 DNA can be artificially modified or in its native state. If recombination into the genome is desired two regions of flanking homology are preferred. Such techniques are generally known for other bacteria, but have been hitherto unsuccessful in P. haemolytica due to tis restriction system.

Vaccines a typically formulated using a sterile buffered salt solution. Sucrose S 15 and/or gelatin may oe used as stabilizers, as is known in the art. It is desirable that the P.

haemolytica vaccines of the invention be administered by the intranasal or intratracheal route, but subcutaneous, intramuscular, intravenous injections also may be used. Suitable formulations and techniques are taught by Kucera U.S. 4,335,106, Gilmour U.S. 4,346,074, and Berget U.S. 4,957,739. Typically, between 10 7 and 10" CFU are administered per dose, although from 10 5 to 10 3 CFU can be used. Adjuvants also may be added.

It will be understood that the term "comprises" (or its grammatical variants) used in this specification is equivalent to the term "includes" and should not be taken as excluding the presence of other elements or features.

EXAMPLES

Example 1 This example demonstrates the isolation and characterization of the type IIs restriction endonuclease PhaI.

FHPMELCD\98121019.4 5 May 1998 (10:58) 9a Bacterium, gzrowth, and crude extract Pasteurella haernolytica serotype 1, strain NADC-D60, was grown 16 hours on 4 Columbia blood agar base plates (lO0mI total volume, Difco, Detroit, MI) without supplemental blood. The cells were harvested in TE (10mM Tris, IntA EDTA, pH pelleted by centrifugation at 16,000 G for 5 minutes at FHPMELCD\98121019.4 5 May 1998 (10:58) WO 95/16045 PCTIUS94/14095 4°C, and washed once in TE. The washed pellet was resuspended in 1.5 ml chromatography running buffer (20mM NaPO 4 10mM 2-mercaptoethanol, pH 4 C) and placed on ice. The bacterial cells were disrupted by sonication for 2 minutes in 15 second bursts. Debris and unbroken cells were removed by centrifugation at 16,000 G for 10 minutes and then filtration of supernatant through a 0.45 um HA membrane. No further treatment of the crude extract was performed prior to chromatography.

Chromatographic separation of proteins All chromatographic procedures were performed at room temperature.

Prepacked heparin-sepharose columns [Econopac heparin columns, Bio-Rad, Richmond, CA] were equilibrated as recommended by the manufacturer. A flow rate of 0.5 ml/minute was used for separation, controlled by 2 HPLC pumps and a controller [Beckman Instruments, Inc, Fullerton, CA]. One ml of crude extract was injected and 10 ml of running buffer was used to wash the column. A linear gradient from 0 to 0.5 M NaCI in 60 ml of running buffer was used to elute proteins. The column was washed with 2M NaCI in running buffer at ml/minute as recommended by the manufacturer, then re-equilibrated to initial conditions of 0 M NaC1 in running buffer prior to additional runs. Fractions ml) were stored on ice prior to activity assay, then frozen at -20 0

C.

Assay for restriction endonuclease activity Aliquots, 5 ul, of the chromatographic fractions were incubated with 1 Al 12 mM MgC1 and 0.25 pg unmethylated bacteriophage lambda DNA (New England Biolabs) at 37°C for 2 hours. After addition of tracking dye, and electrophoresis on a 1% agarose gel in TBE buffer, the banding patterns were visualized by ethidium bromide staining and UV illumination. The active fractions (6ml) were pooled, concentrated 10-fold on 30,000 MW cutoff ultrafilters, and brought to final concentrations of 150 mM NaCI, 10 mM NaPO 4 0.1 mM EDTA, mM 2-mercaptoethanol, 0.25 jig/ml BSA, and 50:50 vol:vol glycerol [pH for storage at I SI~BBR~%II~P~IP~APr~~ wn 95/1604d5 PCT/US94/14095 -11- Determination of the recognition sites for Pha I The recognition sequence was identified using digestion of pBluescript (Stratagene, LaJolla, CA), which resulted in 4 fragments of approximate size 1476, 1057, 252, and 184 base pairs. Double digestion with PhaI and either XhoI or SacI, which cut at opposite ends of the polylinker, showed that one PhaI site mapped at approximately nucleotide 1245, and another at 2735. Additional double digestions with Avall, BglI, Drall, PvuI and Scal were used to map the remaining 2 PhaI sites at approximately nucleotides 2300 and 2490, consistent with the sequences 5'-GATGC-3' and 5'GCATC-3'. Further confirmation was made with PhaI digests of cOX174 and pUC19 DNA, and by sequencing pBluescript PhaI fragments filled in and cloned into pBluescript. Single-stranded $X174 DNA was digested to determine if Phal has activity on this substrate.

Determination of the cleavage sites for Pha I The cleavage site was identified by digestion of a primed-synthesis reaction on pBluescript derivatives (Brown et al. (1980) J. Mol. Biol. 140:143-148). An oligonucleotide containing the PhaI site was annealed and ligated with Sma Icleaved pBluescript SK+ and SK-DNA. Single-stranded DNA containing each orientation was selected and used for the template. Four standard dideoxy DNA sequencing reactions were performed for each template with an appropriate primer.

Additional reactions containing no dideoxy terminator were extended through the PhaI site with the Klenow fragment of DNA polymerase I using 32 P-endlabelled primer with both templates. The extension reaction was stopped by chloroform extraction followed by ethanol precipitation. PhaI or Sfa NI endonuclease was added to the additional reactions and allowed to digest the DNA for 2 minutes.

The reaction was stopped by addition of gel loading buffer and heating to 800C for 3 minutes.

A new restriction endonuclease, PhaI, an isochizomer of SfaNI (Roberts (1990) Nucl. Acids Res. 18 (Suppl.), 2331-2365), was isolated from Pasteurella haemolytica serotype 1, strain NADC-D60, obtained from pneumonic bovine lung.

PhaI recognizes the 5 base non-palindromic sequence 5'-GCATC-3' and IplQ ~IB~Prm4-c- 1 I WO 95/16045 PCTIUS94/14095 -12- 5'-GATGC-3'. Cleavage occurs five bases 3' from the former recognition site and nine bases 5' from the latter recognition site.

Under our experimental conditions, endonuclease activity was eluted from heparin-sepharose columns by 275 to 325 mM NaC1. A single pass through these columns was sufficient to allow identification of both the DNA recognition specificity and cleavage site. Approximately 5000 units of PhaI per gram of wet cells were recovered. In contrast to SfaNI, optimal conditions for PhaI digestion required NaC1 or KC1 concentrations below 50 mM; >50% reduction in activity was observed at the 100 mM NaCl optimum of SfalNI.

Digests of pBluescript resulted in 4 fragments of approximate size 1476, 1057, 252 and 184 bp. Double digestion with PhaI and either Xhol or SacI mapped 2 PhaI sites, one at approximately nucleotide 1245, and another at 2735 of pBluescript. Additional double digestions with PhaI and each of AvaIl, BglI, DraI, PvuI, or Scal mapped the remaining 2 PhaI sites at approximately nucleotides 2300 and 2490, consistent with the sequences 5'-GATGC-3' and GCATC-3'. Digests of pUC19, and cX174 confirmed the recognition specificity of 5'-GCATC-3', which is the same as that of SfaNI. Double digests of pBluescript with Phal and SfaNI resulted in patterns identical to those using either enzyme alone. DNA containing the recognition sequence 5'-GATGC-3' cut 9 nucleotides 5' to the end of the recognition site with both Phal and SfaNI. (Figure 1, lanes 1 and 2) DNA containing the recognition sequence 5'-GCATC-3' cut nucleotides 3' to the end of the recognition site with both PhaI and SfaNI. (Figure 1, lanes 3 and 4) GCATCNNNNN 4 NNNN...3' CGTAGNNNNN NNNN t These data confirm that PhaI is a true isoschizomer of SfaNI. Phal like SfaNI is a type Us enzyme (Roberts, Nucleic Acids Res. 18:2331-2365 (1990)). The type Is restriction enzymes, like the more common type II restriction enzymes, recognize specific sequences and cleave at predetermined site's. Type Is enzymes, however, neither recognize palindromic sequences nor cleave internally to the recognition sequence (Szybalski, Gene 100:13-26 (1991)).

I _Ml k~L~rr- l~ l I WO 95/16045 PCT/US94/14095 -13- Example 2 This example demonstrates the molecular cloning of PhaI endonuclease and methyltransferase.

Cosmid Library Construction High-molecular weight DNA for cosmid cloning was prepared by the large scale DNA isolation method described for gram-negative bacteria in Ausabel et al.

(Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY, NY (1987)). Approximately 100 1g of P. haemolytica strain genomic DNA was digested with 100U of ApoI in NEB buffer #3 at 50°C for 10 minutes. Following digestion, the DNA was phenol-chloroform extracted and ethanol precipitated. The DNA was resuspended in 100 /l TE and layered onto a linear gradient of 10-40% sucrose (Schwartz-Mann Ultrapure) in mM Tris HC1, 1 mM EDTA, 100 mM NaCI, pH 8.0. After centrifugation in a SW40 (Beckman Inst.) at 20,000 RPM for 20 hr, gradient fractions were collected and restriction fragments of approximately 30 kb in length were ligated into Eco RI-digested calf alkaline phosphatase-treated cosmid vector pLAFRX (Ausabel, supra). A standard ligation mixture contained 1 Mg vector, 3 jg P.

haemolytica DNA and 5 Weiss U of T4 ligase in a volume of 10 Ml. The ligation mixture was incubated at 10"C for 16 hr. The DNA was packaged using Promega packaging extract (Promega, Madison, WI) according to the manufacturers' recommendations. E. coli HB101 transduced with the recombinant cosmid library were plated on 2XYT plates containing 10 Ag/ml tetracycline. Cloning efficiencies were approximately 104 recombinant colonies per gg of genomic DNA.

Cloning of PhaI endonuclease and methyltransferase gene Approximately 1 /g of the recombinant P. haemolytica cosmid library was digested with PhaI restriction enzyme. The digested DNA was phenol-chloroformisoamyl alcohol-extracted, ethanol precipitated, and resuspended in TE buffer.

The DNA was electroporated into E. coli AP1-200-9 (Piekarowicz et al., Nucl.

Acids Res. 19:1831-1835 (1991)) and the cells were plated on. LB-broth plates containing 20 ig/ml tetracycline and 35 pg/ml Xgal. The transformed cells were incubated at 42 0 C for 18 hours and transferred to 30°C for 4 hours. The cells I p-pa pi rl 4 sql WO 95/16045 PCT/US94/14095 -14were moved again to 42 0 C and blue colonies, indicating the presence of a cloned methyltransferase gene, were isolated and analyzed. The colonies were screened for restriction endonuclease activity by the technique of Schleif (Method in Enzymology, vol. 65, part I, pp. 19-23 (1980)). Double-stranded DNA mini-preps isolated from restriction endonuclease-positive colonies were analyzed for resistance to digestion by Phal. Recombinant colonies resistant to Phal digestion were presumed to contain a PhaI methyltransferase gene. Cosmid DNA from these cells was electroporated into E. coli DH10B (BRL, Gaithersburg, Maryland) and the cells were plated on LB-broth plates containing 20 tg/ml tetracycline. The transformants containing the PhaI methyltransferase gene were designated E. coli strain PhalMtase.

After digestion with PhaI and transformation of AP1-200-9 strain of E.

coli, fifteen cosmid clones of P. haemolytica genomic DNA were tested for endonuclease activity. The nine clones which were endonuclease-positive were tested for Phal methyltransferase activity. All nine expressed methyltransferase activity in addition to endonuclease activity, as evidenced by resistance to digestion by PhaI of genomic DNA recovered from transformed E. coli. The selective recovery of clones containing functional methyltransferase was due to previous digestion of the cosmid library with PhaI prior to transformation of E. coli.

Recovery of clones containing both PhaI endonuclease and methyltransferase activity is not surprising since restriction and modification enzymes have previously been shown to be closely linked (the proximity of such genes has obvious implications to gene inheritance and the survival of the organism). The AP1-200-9 strain of E. coli (used to screen the cosmid library in this experiment) was designed by Piekarowicz et al., to give color selection for DNA-modifying enzymes (genes). The mrr and mcr systems, with a temperature-sensitive phenotype, induce inducible locus of the SOS response allows for color selection.

All the transformants were blue after incubation at the permissive temperature for the mcr/mrr systems. Recovery of clones containing both Phal endonuclease and methyltransferase activity is not surprising since restriction and modification enzymes have previously been shown to be closely linked (the proximity of such s BI WO 95/16045 PCT/US94/14095 genes has obvious implications to gene inheritance and to the survival of the organism). (Wilson et al., Annu. Rev. Genet. 25:585-627 (1991).) Example 3 This example demonstrates the construction and methylation of a hybrid shuttle vector for introduction of DNA to P. haemolytica.

The following hybrid DNA construct was generated during attempts to introduce site-directed mutations into P. haemolytica. The aroA gene of P. haemolytica, contained on a Hindm-AccI fragment of genomic DNA from strain NADC-D60, was ligated into the Hindm-AccI site of pBluescript. A 700 bp fragment was excised from the coding region of the aroA gene by double digestion with NdeI and StyI. Following digestion, the fragment ends were made blunt by treatment with the Klenow fragment of E. coli polymerasel and deoxynucleoside triphosphates. The deleted plasmid was excised from a 1% agarose gel and electroeluted. The eluted DNA, designated pPhaaroA?, was phenol-chloroform extracted and ethanol precipitated. The fragment was resuspended in TE buffer and ligated with the CmR gene isolated from pBR325. The CmR gene was excised from pBR325 by double digestion with Aat II and ClaI and the fragment was made blunt and purified by the above methods. The CmR fragment ligated with pPhAaroA2 was given the designation pPhAaroACm". Transformation ofE. coli DH10B with pPhAaroACmR conferred CmR to the bacterium.

The hybrid plasmid pPhAaroACmpD80 was constructed by ligating Smal digested pPhAaroACm" with Seal digested pD80 (4.2 kb amp R plasmid from P. haemolytica serotype 1 strain NADC-D80). The resultant hybrid plasmid, approximately 11 kb in size, contained a ColEl and P. haemolytica ori, ampR, and CmR.

For methylation, the hybrid plasmid was electroporated into E. coli strain with or without a cosmid containing cloned PhaI methyltransferase gene.

Plasmid DNA was isolated and purified by CsC1 gradient centrifugation. Phal methyltransferase-treated hybrid plasmid was electroporated into P. haemolytica strain NADC-D60 and then was.reisolated by the above procedures. Plasmid DNA was reisolated from an ampicillin-resistant P. haemolytica transformant by WO 95/16045 PCT/US94/14095 -16the above procedures. The isolated plasmid DNA was tested for resistance to Phal digestion as shown in Figure 2.

Example 4 This example demonstrates that methylated DNA, but not unmethylated DNA, is able to transform P. haemolytica.

Pasteurella haemolytica strain NADC-D60 was grown in 250 ml Columbia broth (Difco) 3 hours at 37 0 C with shaking to late logarithmic phase. The bacteria were centrifuged at 5000 G 15 minutes and the pellet resuspended in 272 mM sucrose at 0°C. The bacteria were washed 4 times in 272 mM sucrose with minute centrifugation at 16,000 G and finally suspended at 50:50 vol:vol packed bacteria:272 mM sucrose on ice. Competent bacteria (100 \tl) were mixed with 1 g hybrid plasmid DNA (harvested from three sources: E. coli DH10B with methyltransferase (PhalMtase); E. coli DH10B without methyltransferase; P.

haemolytica NADC-D60) in 3 separate 1mm electroporation cuvettes (Bio-Rad), plus a fourth no-DNA control. The cells were quickly electroporated after addition of DNA (Bio-Rad Gene pulser) at 1500 V, 800 ohm, 25 uFd with resultant time constants ranging from 7.8 to 8.9 msec. Columbia broth (Iml, 0°C) was immediately added to the electroporated cells and the suspension was kept on ice approximately 10 minutes. The electroporated cells were allowed to recover at 37 0 C with gentle shaking for 1 hour, then broth containing 20 Yg/ml ampicillin was added to bring the final ampicillin concentration to 10 ug/ml and the cells were incubated an additional hour at 37 0 C with shaking. Ten-fold dilutions were plated in duplicate onto blood agar plates containing 5% bovine blood and ug/ml ampicillin. Undiluted cells electroporated with hybrid plasmid obtained from E. coli containing PhaI methyltransferase were plated on 2 tg/ml chloramphenicol after the first hour of recovery. Colonies were enumerated after overnight incubation at 37 0 C and representative colonies were checked for plasmid content.

Hybrid plasmid (pPhAaroACm'pD80) passed through E. coli containing PhaI methyltransferase in a cosmid was able to transform P. haemolytica serotype 1. The hybrid plasmid was stably maintained through multiple passages under WO 95/16045 PCT/US94/14095 -17selective pressure. Whereas DNA not exposed to PhaI methyltransferase was unable to transform P. haemolytica, DNA methylated by PhaI methyltransferase in E. coli yielded 103 transformants per ug plasmid (Table Plasmid DNA passed through P. haemolytica yielded 10S transformants per /g plasmid. This experiment demonstrates that the restriction-modification system of PhaI is important for introduction of exogenous DNA into P. haemolytica serotype 1.

The plating efficiency of transformants was 2 logs lower on chloramphenicol than on ampicillin. All transformants recovered, however, were resistant to both ampicillin, and chloramphenicol upon passage.

The possibility that a system similar to E. coli mcr, mrr, is active in P.

haemolytica was investigated by passage of pPhAroACmD80 through E. coli strain GM2163 previously transformed with the recombinant cosmid containing PhaI methyltransferase (Raleigh et al., Proc. Natl. Acad. Sci. 83:9070-9074 (1986)). Since strain GM2163 is dam-, the resultant DNA would only be modified at PhaI sites (Marinus et al., Mol. Gen. Genet. 192:288-289 (1983)). Efficiency of transformation with this DNA, however, was not substantially different than that using DNA obtained from PhaI Mtase which is dam-methylated (Table It is possible a second restriction system, not readily detectable in cell extracts, is active in P. haemolytica Al. Genes have been described in Neisseria gonnorhea MS 11 which encode for restriction enzymes which are expressed at levels too low to detect biochemically (Stein et al., J. Bact. 74:4899-4906 (1992).

WO 95/16045 PCT/US94/14095 -18- Table 1. Transformation efficiency of P. haemolytica NADC-D60 with hybrid plasmid pPhAaroACm'pD80 purified from various sources'.

Source of DNAb AmpR Cm transformantsd transformantsO CFU/pg DNA CFU/pg DNA E. coli DH10B 0 nd' E. coli PhaIMtase Ixl10 E. coli GM2163 5x10 2 nd P. haemolytica NADC-D60 1x10 5 nd One pg DNA introduced by electroporation using same competent cell preparation.

b Purified by CsCl-EtBr gradient centrifugation.

c Colonies on plates containing 10 pg/ml ampicillin, cells recovered 2 hours prior to plating.

d Colonies on plates containing 2 pg/ml chloramphenicol, cells recovered 1 hours prior to plating.

Not done.

This experiment demonstrates that the restriction-modificai'on system of PhaI plays an important role in the difficulties researchers ha ,,'.fo-,ntered in their attempts to introduce exogenous DNA into P. haemolytica serotype 1.

Protection against PhaI activity may allow genetic manipulation of this organism, which could lead to dramatic improvements in our understanding of pathogenesis and control of pneumonic pasteurellosis in cattle.

Example This example demonstrates the molecular cloning and sequencing of P. haemolytica aroA.

Cloning of P. haemolytica arA. Restriction fragments of P. haemolytica genomic DNA were fractionated by agarose gel electrophoresis. The fragments were probed for homology to a 1.3 kb E. coli aroA fragment by Southern analysis. Under conditions of low stringency, a 3.2-kb HindHI fragment of P.

haemolytica genomic DNA hybridized with radiolabeled E. coli aroA (Fig. 3).

The HindIII fragment was isolated from an agarose gel by electroelution and it was WO 95/16045 PCT/US94/14095 -19cloned into HindIII digested pSK. The recombinant plasmid, pPharoAl, bearing P. haemolytica aroA was identified by complementation of E. coli aroA mutant AB2829 on M9 minimal media containing ampicillin. A Cla I, HindIm double digest of pPharoAl generated a 2.2-kb fragment which was cloned into the AccI and Hindm sites of pSK- giving rise to pPharoA2. Plasmid pPharoA2 also complemented growth of E. coli AB2829 on M9 minimal media. This plasmid was used to determine the sequence of P. haemolytica aroA.

Southern Blotting and Molecular Cloning of P. haemolytica aroA Methods. P. haemolytica genomic DNA was prepared by the method for isolating DNA from gram-negative bacteria. Southern blotting of P. haemolytica restriction fragments fractionated by electrophoresis on a 0.75% agarose was performed as described previously. Blots were hybridized with a radioactively labeled 1.3-kb E. coli aroA fragment. The aroA probe was amplified (Gene-AMP by Perkin Elmer Inc., Branchburg, from E. coli X-L1 Blue (Stratagene, Inc. S.D. CA) genomic DNA using PCR. The primers: TTCATGGAATCCCTTGACGTTACAACCCATC-3' and 5'-AGGCTGCCTGGCTAATCCGCGCCAG-3' used in the PCR reaction hybridize with E. coli aroA nucleotides -3 through 28 and 1308 through 1323 respectively.

The primers were synthesized using an Applied Biosystems oligonucleotide synthesizer (Applied Biosystems Inc.) by the Nucleic Acids Facility, Iowa State University (Ames, IA). DNA was radiolabeled with [a- 32 P] dCTP using a random priming kit (Boehringer Mannheim Biochemicals, Indianapolis, IN). Nylon membranes (Hybond-N, Amersham Corp., Arlington Heights, IL) were incubated with hybridization solution 5X SSC (1X SSC is 0.15M NaCI and 0.015M sodium citrate), 5X Denhardts solution (Maniatis, Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982)), 0.1% SDS, 10 CIg/ml sonicated salmon sperm DNA, containing 1x10 7 CPM of 32 P-labeled probe and formamide at 42°C. After hybridization for 18 hours, membranes were washed twice with 1X SSC and 0.1% SDS for 10 minutes at RT and two times with 1X SSC and 0.1% SDS buffer at 42 0 C for 15 minutes. Membranes were exposed to X-AR (Eastman Kodak Co., Rochester, NY) at -80°C for 24 hours. A positive WO 95/16045 PCT/US94/14095 signal corresponding to a 3.2-kb HindIII fragment of P. haemolytica chromosomal DNA was identified.

HindIII digested P. haemolytica DNA fragments ranging from 3.0-3.4 kb in length were electroeluted from a 1% agarose gel. The HindIl genomic fragments were added to HindII digested alkaline phosphatase treated pSK-vector (Stratagene, Inc. S.D. CA) and ligated overnight at 10 0 C with T4 ligase (BRL, Gaithersburg, MD). The ligation mix was diluted 1:10 with distilled water and electroporated using a Gene Pulser (Bio-Rad Laboratories, Richmond, CA) into E. coli aroA mutant AB2829 (Pittard et al., J. Bact. 91:1494-1508 (1966)). A recombinant plasmid, pPharoAl, complemented AB2829 grown on M9 minimal media containing phosphate buffer, 1 mM MgSO 4 0.1 mM CaC12, 0.2% glucose, thiamine 10 pgiml, 1.5% Noble agar (Difco) and 50 /g/ml ampicillin (Ausubel et al., supra). A Clal, HindIII double digest of pPharoAl produced a 2.2 kb fragment which when cloned into the AccI, HindII sites of pSK- gave rise to pPharoA2. The recombinant plasmid, pPharoA2, which also complemented growth of AB2829 on minimal plates, was used to sequence P. haemolytica aroA.

Nucleotide sequence of P. haemolytic aroA. The nucleotide sequence and the deduced amino acid sequence of P. haemolytica aroA are shown in Fig. 4 and SEQ ID NOS: 1 and 2. An open reading frame of 1302 bases with a coding capacity of 434 amino acid residues was discerned. The deduced molecular weight is 47,296 and the G+C content of the aroA coding region is 43%. The predicted amino acid sequence of P. haemolytica aroA showed 75, 70, 69, and 68% identity with Pasteurella multocida, Klebsiella pneumoniae, Yersenia entercolitica, and Escherichia coli, respectively.

P. haemolytica aroA, like P. multocida aroA (Homchampa et al. Molec.

Microbiol. 23:3585-3593 (1992)), appears to be transcribed from its own promoter. This differs from the usual genetic arrangement in gram-negative bacteria where aroA and serC constitute an operon with aroA distal to the promoter. Evidence to support this claim are the findings that: the nucleotide sequence upstream of aroA on clone pPharoA2 shows no homology with serC genes and complementation of E. coli AB2829 by P. haemolytica aroA WO 95/16045 PCT/US94/14095 -21contained on the 2.2 kb fragment is independent of the fragment's orientation on the cloning vector.

DNA sequencing and Analysis. DNA sequencing was done by the dideoxy nucleotide termination method with single or double stranded templates using the Sequanase 2.0 kit (United States Biochemicals, Cleveland, OH). A series of ordered deletions were made in P. haemolytica aroA contained on pPharoA2 using an Erase-a-base kit (Promega Corp. Madison, WI). Gaps in the sequence were completed using DNA primers synthesized by the DNA core facility at Iowa State University (Ames, IA). DNA sequence analysis was done with MacDNASIS Pro (Hitachi Software Ltd., San Bruno, CA) and MacVector (Kodak Co., New Haven, CT) software.

Example 6 This example demonstrates the construction of a defined P. haemolytica aroA mutant.

Construction of a P. haemolytica aroA mutant. The deletion plasmid, pPhAaroACmR (Table was constructed from pPharoA2 as described above and amplified in E. coli containing a cosmid clone carrying the PhaI methyltransferase gene on a 20-kb P. haemolytica DNA fragment. Although resistant to PhaI endonuclease digestion, introduction of pPhaaroACmR into P. haemolytica strain NADC-D60 by electroporation failed to generate Cm resistant colonies. The inability to transform P. haemolytica with pPhA.aroACmR suggested that plasmids containing a ColE1 origin do not replicate in this bacterium.

WO 95/16045 I"CIVUS94/14095 -22- Table 2. Bacterial strains and plasmids used Strains Characteristics Source/Reference E. coli AB2 829 XL 1-Blue P. haemolytica NADC-D 60 NADC-D7 Q NAflC-DIO Pla.amicds pSK pBCSK pD7 0 pPharoAl pPharoA2 pPharoA3 pPhAaroACMR pPhAaroACmRpD8O php

R

pPharoAAmpR pPharoAAmp'pD7 0 K-12 strain with mutation in aroA Cloning strain used in this work Strain used for DNA sequencing Serotype 1 plasmidless Serotype 1 containing pD7O Serotype 1 containing pD8O cloning vector (Am~pR) cloning vector (CMR) 4.2 kb plasmid encoding streptomycin

R

4.2 kb plasmid encoding ApR 3.2 kb HIndIII fragment containing P. haemoiytica aroA (pSX) HindIII CZaI digest of pPharoAl resulted in 2.2 kb aroA fragment same insert as pPharoA2 on pBCSK StyI tdeI digest of pPharoA2 CmR fragment inserted into deletion site SinaI digested pPhAaroACmR joined to ScaI digested pDSO 2.2 kb Sau 3A fragment of pD8O cloned into pBCSK Axnp fragment of pD8O inserted into unique NdeI site of pPharoA3 HindIII digested pPharoA* AxnpR joined to HindIll digested Pittard and Wallace (1966)

BRL

Stratagene NADC/R. Briggs NADC/R. Briggs NADC/R. Briggs Stratagene Stratagene NADC/R. Briggs NADC/R. Briggs This work This work This work This work This work This work This work This work WO 95/16045 PCTIUS94/14095 -23- Since we have shown that the PhaI methylated hybrid plasmid consisting of plasmids pPhAaroACmR joined with P. haemolytica pD80 (AmpR) could be used to transform P. haemolytica strain NADC-D60 (see above), we investigated the possibility that aroA mutants might arise after transformation with the hybrid plasmid by recombination with the genomic copy of the aroA gene, i.e., "replacement" of the gene. P. haemolytica harboring the hybrid plasmid pPh aroACmpD80 were passed for 100 generations in Columbia broth without antibiotics and plated onto blood-agar plates. The colonies were then replica plated onto blood-agar plates containing 5 pg/ml ampicillin. All colonies had an AmpR phenotype, suggesting that the plasmid was stable in P. haemolytica. This was confirmed by Southern blot analysis which showed that intact plasmid was present in all the AmpR colonies that were analyzed.

Because the number of P. haemolytica transformants generated with hybrid plasmid pPhAaroACmpD80 (AmpRCmR) was 100-fold greater with plasmid isolated from P. haemolytica (10 s CFU/ig DNA) than from E. coli containing the PhaI methyltransferase gene (see above), we reasoned that a replacement plasmid isolated from P. haemolytica would be resistant to enzymatic digestion upon reintroduction into P. haemolytica, and thus more likely to give rise to mutants via homologous recombination. The improved efficiency is presumed to be the result of DNA modifications in P. haemolytica in addition to that of PhaI methylation.

Wih this in mind, hybrid plasmid pPhAaroACmRpD80 was isolated from P. haemolytica strain NADC-D60 and CsC1 purified by the methods described previously. The hybrid plasmid was digested with HindlI and XbaI to produce two fragments consisting of pD80 and pPhaaroACm. Linear deletion plasmid, pPhAaroACnr', was isolated by electroelution and purified using "GlassMax" beads (BRL, Gaithersburg, MD). Approximately 5 txg of the linear plasmid was electroporated into P. haernolytica NADC-D60. The cells were recovered in 1 rm Columbia broth and shaken at 37" C for 1 hour prior to plating on Bloor-agar plates containing 10 pg/mi chloramphenicol. No CmR colonies'were detected after incubation at 37°C for 48 hours. However, this result was not totally unexpected WO 95/16045 PCT/US94/14095 -24since there have been few reports of the successful establishment of linear DNA into bacteria.

Five yg of linearized pPhAaroACmR, isolated from P. haemolytica, was treated with Klenow and deoxynucleoside triphosphates to produce blunt ends.

The DNA was then ligated with T4 ligase overnight to form a circular replacement plasmid. The plasmid was phenol chloroform extracted, ethanol precipitated, resuspended in distilled water, and reintroduced into P. haemolytica by electroporation. The cells were transferred to Columbia broth and allowed to recover for 1 hour. The cells were spread on blood-agar plates containing antibiotic and incubated at 37 0 C for 48 hours. This experiment also failed to generate CmR P. haemolytica colonies.

Additional efforts to produce an aroA mutant resulted in construction of a new replacement plasmid in which aroA was insertionally inactivated by the P. haemolytica f-lactamase gene. This antibiotic resistance cassette was chosen to select gene replacement candidates because we had found that survival of P. haemolyrica transformed with pPhAaroACm'pD80 was approximately 100-fold greater (103 CFU/ItgDNA) on blood-agar plates containing ampicillin than on blood-agar plates containing chloramphenicol.

Molecular cloning of P. haemolytica -lactamase gene was done as follows.

Purified pD80 was patially digested with Sau3A, phenol-chloroform extracted, and ethanol precipitated. The fragments were resuspended in T.E. and ligated overnight into BamHI-digested pBCSK (Stratagene Inc., La Jolla, CA). The ligated mixture was diluted 1:10 with water and electroporated into E. coli The cells were recovered in 1 ml i&C for 1 hour and spread on LB-plates containing 50 gg/ml ampicillin and 20 /4g/ml chloramphenicol.

Restriction enzyme analysis on plasmid isolated from an ampicillin, chloramphenicol resistant E. coli clone revealed a 2.2 kb P. haemolytica insert in pBCSK. This plasmid was designated pPhAmp

R

To demonstrate that pPhAmpR did not possess the pD80 origin of replication, the plasmid was amplified in E. coli DH10B which also contained the PhaI methyltransferase clone Plasmid pPhAmp was isolated from E. coli as described previously, CsCI purified and introduced PP IPIIPL~bLIIL IIBmBLPsullmr~P- r~ a WO 95/16045 PCTIUS94/14095 into P. haemolytica by electroporation. Since this plasmid did not confer ampicillin resistance to P. haemolytica strain NADC-D60, we concluded that the antibiotic resistant fragment did not contain the pD80 origin of replication and that the fragment encoding the -lactamase gene could be used to construct a deletion plasmid.

Construction of the deletion piasmid involved the following. The 3lactamase gene was excised from pPhAmpR by HindII, Xbal digestion and treated with Klenow and deoxy-ribonucleotides to generate blunt ends. The 3-lactamase gene was ligated into the Klenow treated unique NdeI site of pPharoA3 (Fig. to produce pPharoA'Amp. Insertional inactivation of aroA on pPharoA'ampR was demonstrated by failure of the plasmid to complement AB2829. Plasmid pPharoA" AmpR was amplified in E. coli DH10B (BRL) which also contained the recombinant cosmid carrying PhaI methylase recombinant cosmid. Although PhaI methylated pPharoA'AmpR was resistant to digestion by PhaI, introduction of this plasmid into P. haemolytica failed to generate ampicillin resistant colonies.

To increase the likelihood of allelic replacement between the deletion plasmid's inactivated aroA and P. haemolytica chromosome, we constructed an aroA mutant-hybrid plasmid consisting of pPharoA'Amp" and a 4.2-kb P. haemolytica plasmid (pD70, which confers streptomycin resistance (Fig.

The SmR plasmid was isolated from P. haemolytica using methods described previously. The strR plasmid was digested at a unique HindIII site and ligated with HindlT digested pPharoA'Amp. The resultant hybrid plasmid, pPharoA- (Fig. was PhaI methyltransferase modified in E. coli containing the cosmid clone of the PhaI methylase gene. The hybrid plasmid was isolated from E. coli, CsCI purified and introduced into P. haemolytica strain by electroporation. The cells were resuspended in Columbia broth for 2 hours at 37 0 C and spread on blood-agar plates containing 10 /g/ml ampicillin.

Transformation efficiency of the hybrid plasmid yielded approximately ampicillin resistant colonies/jg DNA. Eight AmpR colonies were grown overnight in Columbia broth containing 1 jg/ml ampicillin. Chromosomal DNAs from the parental strain and from the AmpR colonies were digested with Hindm and probed L_ 9 I I -I -r WO 95/16045 PCT/US94/14095 -26by Southern blotting with P. haemolytica aroA, pBCSK, and pD70. The results indicated that intact pPharoA-Amp'pD70 was present in the AmpR colonies.

Eight Amp' clones were grown overnight in Columbia broth containing 1 p/g/ml ampicillin. Chromosomal DNAs from the parental strain and from the Amp' clones were digested with HindII and analyzed by Southern blotting with P. haemolytica aroA, pBCSK, and pD70 radio-labeled probes. The results indicated ihat intact pPharoA-AmppD70 was present in the Amp' clones (data not shown). The eight AmpR cultures were transferred to Columbia broth containing 1 Ag/ml ampicillin and cultured at 370 C. The bacteria were transferred to fresh media daily and this process was continued for approximately 100 generations.

The eight cultures were streaked for isolation without antibiotic selection and a single colony of each was passed into Columbia broth containing either 1 yg/ml ampicillin or 1 /g/ml chloramphenicol. Two of the eight survived on the broth containing ampicillin, none on chloramphenicol. Passage from ampicillin broth onto blood-agar plates containing either ampicillin or chloramphenicol or streptomycin confirmed the two clones were AmpR, Cms, Sm s Also the two AmpR clones were spread onto plates of chemically-defined medium for P.

haemolytica cultivation (Wessman, Applied Microbiol. 14:597-602 (1966)). This medium lacks the aromatic amino acid tryptophan. The parent strain grew on the defined medium but the AmpR clones did not. Upon addition of tryptophan to the defined medium, growth of the AmpR clones was comparable to that of the parent strain. The E. coli aroA mutant AB2829 also required tryptophan to grow on the chemically-defined medium for P. haemolytica cultivation. DNAs from the two colonies possessing AmpR, Cms, SmS,aroA' phenotypes were analyzed by Southern blotting. The results indicated that both had insertionally inactivated aroAs.

Moreover, Southern blotting also confirmed that both pD70 and pBCSK sequences were no longer present in the aroA mutants (Figure 6).

Construction methods for P. haemolytica mutants. The 4.2 kb ampicillin resistance encoding plasmid of P. haemolytica (pD80) was partially digested with Sau3A and ligated into the BamHI site of pBCSK* (CmR) (Stratagene Inc., La Jolla, CA). The ligation mix was diluted 1:10 in distilled water and electroporated sL~slb*~ I WO 95/16045 PCT/US94/14095 -27into E. coli DH-10B (BRL, Gaithersburg, MD). After recovery in 1 ml SOC at 37 0 C, the cells were spread onto B-agar plates containing 50 pg/ml ampicillin.

Plasmid, pPhAmR, contained a 2.2-kb P. haemolytica fragment which imparted ampicillin resistance to E. coli to up to 100 yg/ml. Plasmid, pPhAmpR, was digested with Hindm and XbaI digestion and the fragment ends were made blunt by incubation with deoxynucleotide triphosphates and the large Klenow fragment of E. coli polymerase I. The fragment encoding ampicillin resistance was electroeluted. P. haemolytica aroA contained on pPharoA3 was digested at an unique restriction site within the coding region of aroA with NdeI and the fragment ends were made bunt as described previously. The fragment encoding ampicillin resistance was blunt-end ligated with T4 ligase into pPharo2 thus generating pPharoA-AmpR. Plasmid pPharoA'AmpR was digested with HindIII and dephosphorylated with calf alaline phosphatase. A 4.2 kb plasmid encoding SmR isolated from P. haemolytica strain NADC-D70 (Chang et al., J. DNA Sequencing and Mapping 3:89-97 (1992)) was also digested with HindIII and the two plasmids were ligated with T4 ligase to generate the hybrid plasmid The hybrid plasmid was electroporated into E. coli Pha IMtase which contained the PhaI methyltransferase gene on cosmid pLAFRX (Ausubel, supra).

P. haemolytica strain NADC-D60 is a plasmidless strain which was isolated from a cow with pneumonic pasteurellosis. The PhaI methylated hybrid plasmid was CsC1 purified and 1 ug plasmid and 30 p1 of P. haemolytica strain NADCwere transferred to an 0.2 cm. cuvette and electroporated at 15,000 volts/cm with 800 ohms. The resultant time constant was approximately 9 milliseconds.

Cells were transferred to 2 ml Bacto Columbia broth (Difco Labs, Detroit, MI) and incubated at 37°C for two hours and spread on Difco Columbia blood-agar plates containing 10 /g/ml ampicillin. Eight ampicillin resistant P. haemolytica colonies were isolated after incubation at 37°C for 18 hours. The colonies were then transferred to Bacto Columbia broth containing 1 /ig/ml ampicillin and incubated at 370C. Daily passage into fresh medium containing 1. /g/ml ampicillin was carried out for three days at which time the cultures were transferred onto Columbia broth blood-agar plates containing 10 Atg/ml ampicillin and incubated at

-M

C

WO 95/16045 PCT/US94/14095 -28- 37°C overnight. The next day, colonies were replica-plated onto Columbia broth blood-agar plates containing 10 pg/ml or 50 gg/ml streptomycin and a chemicallydefined medium for P. haemolytica cultivation (Wessman, supra). The defined medium contains 15 amino acids and includes the aromatic amino acids phenylalanine and tyrosine but not tryptophan. The clones unable to grow on the chemically-defined medium for P. haemolytica cultivation were presumed to be aroA'. Genomic DNA isolated from colonies with AmpR, Cms, Sm s aroAphenotypes were analyzed by Southern blotting. Southern blotting was performed as described previously with the exception that after hybridization the membranes were washed twice for 10 minutes each in Ix SSC and 0.1% SDS at 420 C and twice more for 15 minutes each in 0.lx SSC and 0.1% SDS at 650 C.

c II ea, -e~I I PCT/US94/14095 wn oII645 -29- SEQUENCE LISTING GENERAL INFORMATION: APPLICANTS: Biotechnology Research and Development Corporation and The United States of America (ii) TITLE OF INVENTION: CONSTRUCTION OF PASTEURELLA HAEMOLYTICA

VACCINES

(iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Banner, Birch, McKie and Beckett STREET: 1001 G Street, N.W.

CITY: Washington STATE: D.C.

COUNTRY: U.S.A.

ZIP: 20001 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE: 06-DEC-1994

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: Kagan, Sarah A.

REGISTRATION NUMBER: 32,141 REFERENCE/DOCKET NUMBER: 48294 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 202-508-9100 TELEFAX: 202-508-9299 TELEX: 197430 BBMB UT INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Escherichia coli (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: AGGCTGCCTG GCTAATCCGC GCCAG INFORMATION FOR SEQ ID NO:2: all a l- la rl I 'PB rrrmbP~- WO 95/16045 PCT/US94/14095 SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Escherichia coli (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: TTCATGGAAT CCCTTGACGT TACAACCCAT C 31 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 1556 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Pasteurella haemolytica (ix) FEATURE: NAME/KEY: CDS 40 LOCATION: 184..1486 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TATGAGGCAT TACTGCGTGA AGGCGTGATT GTTCGCTCGA TAGCAGGTTA TGGAATGCCG AATCATTTAC GCATTAGTAT GCCTTTACCG CAAGAAAACG AGAGATTTTT TACTGCCTTA 120 TTGAAAGTGT TAGCTTAACA AGCGGTTACC TTTTATGAAA ATTTTACAAA TTTAAAGAGA 180 AAA ATG GAA AAA CTA ACT TTA ACC CCG ATT TCC CGA GTA GAA GGC GAG 228 Met Glu Lys Leu Thr Leu Thr Pro Ile Ser Arg Val Glu Gly Glu 1 5 10 ATC AAT TTA CCT GGT TCT AAA AGC CTG TCT AAC CGA GCC TTA TTA TTA 276 Ile Asn Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg Ala Leu Leu Leu 25 GCC GCC TTA GCC ACC GGT ACG ACT CAA GTG ACC AAT TTA TTA GAT AGT 324 Ala Ala Leu Ala Thr Gly Thr Thr Gln Val Thr Asn Leu Leu Asp Ser 40 GAT GAT ATT CGA CAT ATG CTC AAT GCC TTA AAA GCG TTA GGC GTG AAA 372 Asp Asp Ile Arg His Met Leu Asn Ala Leu Lys Ala Leu Gly Val Lys 55 PCT/US94/14095 WO 95/16045 PTU9/49 TAT GAG Tyr Giu CTA TCG GAC GAT AAA ACC GTC TGT GTA CTT GAA GGG ATT GGT Leu Ser Asp Asp Thr Val Cys Vai Leu Giu Gly Ile Gly

GGA

Gly

GGC

Gly GCT TTT AAG GTT Ala Phe Lys Val ACG GCA ATG Thr Ala Met

GAA

Glu

CCG

Pro

CAG

Gin

GTT

Val 160

TTT

Phe

AAA

Lys

ATT

Ile

TAT

Tyr 145

TGC

Cys

CTA

Leu

TCC

Ser

AAA

Lys 130

TTA

Leu

AGG

Arg

ACC

Thr

CAA

Gin 115

CAC

His

GAA

Glu

GGC

Giy

GCA

Ala

CGA

Arg 100

ATC

Ile

TTA

Leu

AAT

Asn

GGA

Gly

TTG

Leu

CAA

Gin 85

CCA

Pro

ATT

Ile

GTC

Val

GAA

Giu

AAA

Lys 165

CTG

Leu

AAC

Asn

CTT

Leu

CTT

Leu

GAT

Asp

GGC

Gly 150

GTG

Val

ATG

Met

GGC

Gly

GCA

Ala

ACC

Thr

GCT

Ala 135

TAT

Tyr

CAA

Gin

TCT

Ser

TTA

Leu

GCA

Ala

GGT

Gly 120

TTA

Leu

CCA

Pro

ATT

Ile

GCC

Ala

TCA

Ser

GCA

Ala 105 Glu

CGC

Arg

CCG

Pro

CT

9

TT

Le cc~ Pr CA2 G1,

TTC

Let 180 GAA ATT GAG ATT ATC GGT GAT Giu

ACC

Thr

TAC

Tyr

GGT

Gly 240

GCC

Ala

TCG

Ser

GCA

Ile

CTT

Leu

AAA

Lye 225

AAT

Aen

TCC

Ser

ATC

Ile

AAA~

Giu

TCG

Ser 210

ACC

Thr

TAT

Tyr

GGT

Gly

CAA

Gin Ile 195

ATG

Met

TTT

Phe

TTG

Leu

GCG

Ala

GGC

Gly 275

ACT

Ile

ATG

Met

TTA

Leu

GTG

Val

ATT

Ile 260

GAC

Asp

TGG

Gly

AAC

Asn

GTT

Val

GAG

245

AAG

Lys

CGC

Arg

GGA

Asp

GAT

Asp

AAA

Lye 230

GGA

Gly

GCA

Ala

TTG

Leu

GAG

CTG GTA Leu Val 200 TTT GGT Phe Gly 215 GGT AAA Gly Lye GAT GC Asp Ala GGT AAA Gly Lys TTT GCC Phe Ala 280 GAT TTT GAC GGC Asp Gi 170 CCA TTAi Pro Leu 185 TCA AAA Ser Lys ATT ACG, Ile Thr CAA GGC GIn Gly TCT TCT Ser Ser 250 GTA ACG Val Thr 265 GAT GTG Asp Val PTT CAA G TTT u Phe 0

TGT

Cye A. AGA o Arg

~GTA

-1 Val

;GCA

Ala 155

TCG

Ser

GCG

Ala

CCT

Pro

GTT

Val

TAT

Tyr 235 GCC '1 Ala1 GGC I Gly I TTG G Leu G GCC G Ala G 3

CTC

Leu

TTA

Leu

ATG

Met

GGG

Gly 140

ATT

Ile

ATT

Ile

GAA

Giu

TAT

Tyr

GAA

Glu 220

;TT

lai ECT 'I er I1 LTT G lie G

AA

lu L.

2 'AG C iu G 00

GGC

Gly

AAA

Lye

AAA

Lys 125

GCA

Ala

AGC

Ser

TCC

Sex

GGC

G ly

P.TT

Ile 205

AAT

ksn

;CT

kla

LAT

'yr

;GT

AA

ys

AA

in

AAT

Asn

GGT

Gly 110

GAA

Giu

GAG

Giu

AAT

Asn

AGO

Ser

GAT

Asp 190

GAT

Asp

CGA

Arg

CCA

Pro

TTC

Phe

AAA.

Lye 270

ATG

Met

TCC

Ser

CAA

Gin 175

ATG

Met

ATT

Ile

GAT

Asp

CAA

Gin

TTA

Leu 255

PAA

Lys

~GG

.CG

?ro 708 756 804 852 900 948 996 1044 1092 1140

GCA

Ala

GAG

Glu

CGC

Arg

OTA

Val

AGC

Ser 468 516 564 612 660 Ala Lys Ile Thr Trp Gly Giu 290 CTA AAA GGC GTA GAT ATG GAT Leu Lye Gly Val Asp Met Asp 305 310 Asp Phe Ile Gin 295 ATG AAT CAT ATT Met Asn His Ile CCT GAT GCG GCA ATG Pro Asp Ala Ala Met 315 WO 95/16045 PCT[US94/14095 ACG ATT Thr Ile 320 AAT ATI Asn Ile GCA ACC Ala Thr GGG GAA Gly Giu CAC GCT His Ala 385 TCA TTA Ser Leu 400 TGT ACC Cys Thr TCG GTC Ser Val -32- GCA ACA ACC GCT TTA TTT GCC GAA GGA GAA ACA GTT ATC Ala Thr Thr Ala Leu Phe Ala Giu Gly Glu Thr Val Ile 325 330 TAT AAC TGG CGG GTA AAA GAA ACC GAC CGC TTG ACA GCA Tyr Asn Trp Arg Val Lys Glu Thr Asp Arg Leu Thr Ala 340 345 350 GAA TTG CGT AAA GTC GGG GCA GAG GTA GAA GAA GGG GAA Glu Leu Arg Lys Val Gly Ala Glu Val Glu Giu Gly Glu 355 360 365 GAT TTT ATT CGG ATT CAA CCG CTT GCG TTA GAA AAC TTC Asp Phe Ile Arg Ile Gin Pro Leu Ala Leu Giu Asn Phe 370 375 380 GAA ATT GAA ACC TAT AAC GAT CAC CGT ATG GCA ATG TGT Giu Ile Giu Thr Tyr Asn Asp His Arg Met Ala Met Cys 390 395 ATT GCG TTA TCG AAT ACA GAA GTG ACG ATC TTA GAT CCA Ile Ala Leu Ser Asn Thr Giu Val Thr Ile Leu Asp Pro 405 410 OCT AAA ACG TTC CCG ACT TAC TTT AGG GAC TTG GAA AAA Ala Lys Thr Phe Pro Thr Tyr Phe Arg Asp Leu Giu Lys 420 425 430 AGA T AAAAGTAAAA AAGGATTCAG AAAACTGAAT CCTTTTTACG Arg

CGC

Arg 335

ATG

Met

GAA

Giu

CAG

Gin

TTT

Phe

A.AT

Asn 415

TTA

Lieu 1188 1236 1284 1332 1380 1428 1476 1526 1556 TTTTATTGTG GCAGACTAAG CCCAACCGCT INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 434 amino acids TYPE: amino acid TOPOLOGY,. linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Giu Lys Leu Thr Leu Thr Pro Ile Ser Arg Val Giu 1 5 10 Asn Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg Ala Leu 25 Ala Leu Ala Thr Gly Thr Thr Gin Val Thr Asn Leu Leu 40 Asp Ile Arg His Met Leu Asn Ala Leu Lys Ala Leu Gly s0 55 Giu Leu Ser Asp Asp Lys Thr Val Cys Val Leu Giu Gly 65 70 Ala Phe Lys Val Gin Aen Gly -Leu Ser Leu Phe Leu Gly 90 Gly Leu Asp Val Ile Asn Giu Leu Ser Lys Gly Ala WO 95/16045 PTU9/49 PCT[US94/14095 Thr Ala Met Lys Ser Gin 115 Ile Lys His 130 Tyr Leu Glu 145 Cys Arg Giy Arg Pro 100 Ile Ile L.eu Vai Asn Giu Giy Lys 165 Leu Leu Asp Gly 150 Val.

Ala Thr Ala 135 Tyr Gin -33 Ala Ala 105 Gly Giu 120 Leu Arg Pro Pro Ile Asp Leu Cys Pro Arg Gin Val Leu Ala 155 Gly Ser 170 Leu Met Gly 140 Ile Ile Lys Lys 125 Ala S er Ser Gly 110 Giu Giu Aen Ser Glu Arg Val S er Gin 175 Giu Pro Gin Val 160 Phe Leu Thr Ala Leu Leu Met Ser Ala Pro Leu Ala Ile Leu Lys 225 Glu Ser 210 Thr Asp Asp Lys Leu Phe 215 Glu Gly Asp 190 Tyr Ile Asp 205 Giu Asn Arg 220 Met Ile Asp Gly Lye Gin Gly Tyr Aen Tyr Leu Val Ser Ile Lys Lys 305 Ile Ile Thr Giu Gly Gin Ile 290 Gly Ala Tyr Giu Asp 370 Ala Gly 275 Thr Val Thr Asn Leu 355 Phe Ile 260 Asp Trp Asp Thr Trp 340 Arg Ile Glu Gly Asp 245 Lys Ala Gly Arg Leu Phe Gly Giu Asp 295 Met Asp Met 310 Ala Leu Phe 325 Arg Val Lye Lys Val Gly Arg Ile Gin 375 Ala Ser Ser 250 Lys Val Thr 265 Ala Asp Val 280 Phe Ile Gin Asn His Ile Ala Giu Gly 330 Giu Thr Asp 345 Ala Giu Val 360 Al a Vai Ala Pro Gin Gly 240 Ser Tyr Phe Leu Ala 255 Gly Ile Gly Leu Ala Pro 315 Glu Arg Glu Glu Lye 285 Giu Gin 300 Asp Ala Thr Val Leu Thr Giu Gly 365 Giu Aen 380 Ala Met Lys 270 Met Ser Ala Ile Ala 350 Glu Phe Cys Lys Gly Pro Met Arg 335 Met Glu Gin Phe Ser Ala Leu Thr 320 Aen Ala Gly His Ser 400 Pro Leu Ala Leu Ala Glu Ile Glu Thr 385 Asn Asp His Arg Met 395 Leu Ile Ala Leu Ser Aen Thr Glu Val 405 Thr 410 Ile Leu Asp Pro Asn Cys 415 Thr Val 420 Arg Phe Pro Thr Tyr Phe Arg 425 Asp Leu Glu Lye Leu Ser 430 WO 95/16045 WO 9516045PC]'/US94/J 4095 ANNEX M3 International Application No: PCT/

MICROORGANISMS

Opto h.oot In Ci., OUO w.woith~ tKg mi~looeisvi taloned to 04 Pops. uto= 2 82& o4 the d..cte4Ioa I A- IOEMT1FICAT11OPI Of DEPOSIT I AMERICAN TYPE CULTURE COLLECTION Aidles 61depoitay inaml" plckedifto poosw c~o and 10vn417) 4 12301 Parki awn Drive Rockville, Maryland 20852 United States of America 0410 of deposit IAce&"h bo& 2 December 1993 (02.12.93) 169499 a. ADDITIONAL INDICATIONS I bJs% Ni ^*Ig *00111ble). This 16n4*ri,,t., is C00VflS.d Onl a&operate aneichod she, P. haemolytica containing a 4.2kb plasmid C. DESIONATIOD ST.*T1A FOR WHICH INDICATIONS ARE MAO& 6 (11 IM. adkoueons ane OW Ie. aA sloold Stalt,) 0. SEPARATE VIIAISHING Of INDICATIONS 0 06410 6441Ak 0 ot spitekable) !I.e tested beloell be oubl~nd to IhS 1n.IftltlA" Buisav L144 4 (Spocty the "eui 04 the kewkca -ACCOGsleft h,.flba at CDeposot-) 1. 0 1h.2 eillat kla Ielee..,lwst £110 iponav" toobCotewoft Mad~4 (to be checked by lSefocerv~mg Omfcs) Inora Y. FRQTS CAi.hotiod Officae C Tb. date *I roccoot (4Othe 1110 e&Cant by Ved InI.,a1~0l" GW101at3 ro. PCTIIOiI34 ij*n.orr 1141) WO 95/16045 PCT/US94/ 14095 ANNEX M3 Interniational Application No: PCT/

MICROORGANISMS

olt"~& Sho.al in co~rcIIon wN1 £114 nmkce..anism tt9ervod le, 96 MQG4-.- i A. IDENTIFICATION Of OE9OUIT I tuhef d#00sila ame IdnaUfted On an oddtlJ..taJ shooeM Her" of de"eltary lnglllU0A AMERICAN TYPE CULTURE COLLECTION Addrles*96061,1r *IyI$0 WlAQ pollcd nlcuiy 12301 Park) awn Drive Rockville, Maryland 20852 United States of America Date el 060cej6 Acc.essl et Nubw4 2 December 1993 (02.12.93) 9n 01. ADDITIONAL INDICATION$U 1 blank It I o&Pyl~cable). Th1.ill nIanaU4A Is Conlined "en 4atl ArEI*Ucb hen E. coli PhaI~tase C. DESIGNATED STATES FOR WHICH INDICATIONS ARK MAO* I (It the4 ltdications file ne t l01 daslonalel; 3lates) 0. SEPARATE FURNISHING Of INDIICATIONS' I blank Nt not applicable) a 14gl n .4n Nlel belt. 1 ble sub"ned It t114 Intaenalvanal Bejeaw taat,I (Specity £1h4 0mal t r ofvI e I. ndC.£tone It. 0TN&s*heel -as onthen ,lsellosl~ l &VPhCAtt*A -h4A f-LaE (0 be £ho(h~d bY £1h. tecen-4 omcs Einora Y. Rivera Dvso pci unemxa 1 ISI (Awthorilod Offk#4 0The elale of rce~fi (iethe1 applkcent) bY th14 telornatlonai alifew to (Authottzed Offiewt) Fati PCTIRO/13I (Ijanea t) 1 (January 1991) WO 95/16045 PCIUS9414095 -36- ANNEX M3 hinernaflonal Appllcallon No: PCT/I

MICROORGANISMS

Oipti.... shoot iA cefncto wit tk mkooaj a r~ro to on .26. 4.ilaon A. 1INTIfFICATION or DEPOSIT'I Furthoc cleeoeas are ident1.d on an .dfc..Ai up,..q No-@ of. depository InstaIt"U~ 4 AMERICAN TYPE CULTURE COLLECTION Addses Of d4.noeary in~~o licuda "se coeaw o" 12301 Parkl1awn Drive Rockville, Maryland 20852 United States of America Data of d6P44I I Access$" N...rbw 2 December 1993 02.12.93 1C51 S1. ADDITIONAL INDICATIONS I (leer, 64ank it oot apollcabl. This Inlormaenen is coMAnori on a aepefoal attached .Ihd Q P. haemolytica C. DESIGNATED STATES FOR WHICH INDICATIONS ARK MADE'1 (111th IN. wilaion ar. 664 1.4 &A d @sl*A*4 Sltae) 0. SEPARATE FURNISHING Of INDICATIONS I (leas blenN 01 eepl~kble) TIe...ds*Ilns kated baeo dA be 9sbmfi Is th NtIVAns~ti Ilurtau Isle( 6 (Specify the *erAfal nhtw. .4 the IWdk&eA 0.g..

-ACccoesnf Nenbal of I..poso E. TN.. .40 f.c...wbIN.,l nlqe eoCd~On rn 01"d Ile be Chocikad by the ecern Oftse) CDTN. dale 01 FreceFI (llm IN. sp#ksnt) by cho Inirrrafteonal (Air~mIsL~- T alnlDvso (A.ANhrtjod O~fcee Ftrm PCTiRO1134 IJ4rreary IMl) (January 1"1fl

Claims (25)

1. A method for producing a mutation in a particular region of DNA of a P. haemolytica genome, comprising the step of: isolating said region of the genome from P. haemnolytica; introducing a mutation into said region to form a mutated DNA region; methylating said mutated DNA region with a methylating enzyme which inhibits endonuclease cleavage at a recognition sequence selecterd from the group consisting of GATGC-3', and 5'-GCATC-3', to form methylated DNA; introducing said methylated DNA into a P. haemolytica cell to form transformants; and screening said transformants for those which have said mutation in said region on chromosomal DNA of said P. haemolytica cell.

2. The method of claim 1 wherein said step of methylating is performed by passage of said DNA region through a methylating cell containing Phal methylase. 15 3. The method of claim I wherein said step of methylating is performed by passage of said DNA region through a methylating cell containing SfaNI methylase.

4. The method of claim 1 wherein the step of methylating is performed in vitro. The method of claim 1 wherein the methylating enzyme is Phal methyltransferase. The method of claim 1 wherein the methylating enzyme is SfaNI methyltransferase.

7. The method of claim 2 wherein said methylating cell is a P. haemolytica strain which contains no PhaI restriction endonuclease activity.

8. The method of claim 2 wherein said methylating cell is a bacterium other than P. haemolytica which contains a gene encoding Phal methylase. FHPMELCD\98121019.4 -5 May 1998 (10:58) IL- l WO 95/16045 PCT/US94/14095 -38-

9. The method of claim 2 wherein said methyating cell is a bacterium other than Streptococcusfaecalis which contains a gene encoding SfaNI methylase. The method of claim 1 wherein said methylated DNA is introduced into P. haemolytica on a plasmid containing a P. haemolytica 4.2 kb StrR plasmid deposited at the ATCC as Accession No. ATCC 69499.

11. The method of claim 10 further comprising: screening said transformants for loss of said 4.2 kb Str plasmid.

12. An isolated and purified gene encoding PhaI methyltransferase.

13. An isolated and purified gene encoding Phal restriction endonuclease.

14. A preparation of PhaI methyltransferase free from PhaI restriction endonuclease. A preparation of PhaI endonuclease free from PhaI methyltransferase.

16. The preparation of claim 14 which is free from all other P. haemolytica proteins.

17. The preparation of claim 15 which is free from all other P. haemolytica proteins.

18. A chimeric plasmid for unstable introduction of genetic material into P. haemolytica comprising two plasmids covalently linked to each other, wherein the first plasmid is a 4.2 kb StrR plasmid of P. haemolytica deposited at the American Type Culture Collection as Accession No. ATCC 69499; and the second plasmid is a plasmid which cannot replicate in P. haemolytica.

19. The chimeric plasmid of claim 18 further comprising: a region of the chromosome of P. haemolytica wherein said region harbors a mutation. A P. haemolytica mutant made by the process of claim 1.

21. P. haemolytica strain NADC-D60aroA', deposited at the ATCC as Accession No. ATCC 55518.

22. A P. haemolytica strain which harbors a mutation which abolishes expression of PhaI restriction endonuclease. a

23. A vaccine comprising an attenuated, live, mutant of P. haemolytica, which comprises an aroA mutation.

24. The vaccine of claim 23 wherein said mutation is a non-reverting mutation. The vaccine of claim 23 wherein said mutation is an insertion mutation.

26. The vaccine of claim 23 wherein said mutation is genetically linked to a selectable marker.

27. A method for producing a mutation in a particular region of DNA of a P. haemolytica genome, comprising the step of: isolating said region of the genome from P. haemolytic a; introducing a mutation into said region to form a mutated DNA region; introducing said mutated DNA region into a P. haemolytica cell which does not express a Ph0 restriction endonuclease, to form transforma'.,; and screening said transformants for those which have said mutation in said region on chromosomal DP'A of said P. haemolytica cell. S 15 28. The method of claim 27 wherein said P. haemolytica cell which does not express a SPhaI restriction endonuclease is a natural isolate.

29. The method of claim 27 wherein said P. haemolytica cell which does not express a PhaI restriction endonuclease is a mutant made by chemical mutagenesis.

30. The method of claim 27 wherein said P. haemolytica cell which does not express a Phal restriction endonuclease is a mutant made by the process of claim 1.

31. A P. haemolytica mutant made by the process of claim 27.

32. An isolated and purified P. haemolytica strain which has been genetically modified .by the stable introduction of DNA.

33. The P. haemolytica strain of claim 32 wherein the introduced DNA has recombined with genomic DNA of P. haemolytica. Biotechnology and Research Development Corporation and The United States of America represented by the Department of Agriculture By their Registered r'Ptent Attorneys FREEHILLS PATENT ATTORNEYS 5 May 1998 FHPMELCD\98121019.4 5 May 1998 (10:58)